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Journal of Plant Research

, Volume 125, Issue 1, pp 185–195 | Cite as

Fast loading ester fluorescent Ca2+ and pH indicators into pollen of Pyrus pyrifolia

  • Haiyong Qu
  • Xueting Jiang
  • Zebin Shi
  • Lianmei Liu
  • Shaoling ZhangEmail author
Technical Note

Abstract

Loading of Ca2+-sensitive fluorescent probes into plant cells is an essential step to measure activities of free Ca2+ ions in cytoplasm with a fluorescent imaging technique. Fluo-3 is one of the most suitable Ca2+ indicators for CLSM. We loaded pollen with fluo-3/AM at three different temperatures. Fluo-3/AM was successfully loaded into pollen at both low (4°C) and high (37°C) temperatures. However, high loading temperature was best suited for pollen, because germination rate of pollen and growth of pollen tubes were relatively little impaired and loading time was shortened. Moreover, Ca2+ distribution increased in the three apertures of pollen after hydration and showed a Ca2+ gradient, similar to the tip of growing pollen tubes. The same protocol can be used with the AM-forms of other fluorescent dyes for effective labeling. When loading BCECF-AM into pollen at high temperature, the pollen did not show a pH gradient after hydration. Ca2+ activities and fluxes had the same periodicity as pollen germination, but pH did not show the same phase and mostly lagged behind. However, the clear zone was alkaline when pollen tube growth was slowed or stopped and turned acidic when growth recovered. It is likely that apical pHi regulated pollen tube growth.

Keywords

CLSM Pollen Fluo-3/AM Temperature BCECF-AM 

Abbreviations

BCECF-AM

3′-O-Acetyl-2′,7′-bis(carboxyethyl)-4 or 5-carboxyfluorescein diacetoxymethyl ester

CLSM

Confocal laser scanning microscopy

[Ca2+]cyt

Cytosolic-free calcium

dd water

Double distilled water

ECFP

Enhanced cyan fluorescent protein

EGTA

Ethylene glycol-bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid

EYFP

Enhanced yellow fluorescent protein

pHi

Intracellular pH

SSR

Shortest significance ranges

Notes

Acknowledgments

We thank Professor Megumi Iwano of Nara Institute of Science and Technology (Japan) who supplied transgenic seed that expressed the YC3.1 gene in pollen grains and papilla cells. We also thank Professor Deng Shaoping in ZheJiang GongShang University who let us freely use the confocal instrument in his laboratory. We thank all the scientists who helped to improve our manuscript. We are indebted to the two anonymous reviewers, whose insightful comments resulted in a much improved final version of the manuscript. This work was supported by the National Natural Science Foundation of China (31071759) and the Scientific Research Fund of Huaiyin Institute of Technology (No: HG0606).

Supplementary material

There are three phases of movie of the same pollen tube. Pollen were cultured in dye-free medium for 2 h in darkness after loading BCECF-AM at high temperature (37°C) for 30 min and then observed with CLSM. Confocal images were collected every 10 min for 3 h. We adjusted field of vision of CLSM every hour, because of movement from pollen tube growth. The first movie (pH 1.mpg) is images collected during the first 0–50 min (mpg 525 kb)

The second movie (pH 2.mpg) is images collected during the middle 60–130 min (mpg 829 kb)

The third movie (pH 3.mpg) is images collected in the last 50 min. These movies reveal the change of pH dynamics at of pollen tube tips during their growth (mpg 423 kb)

10265_2011_440_MOESM4_ESM.doc (101 kb)
Supplementary material 1 (DOC 103 kb)
10265_2011_440_MOESM5_ESM.doc (10.3 mb)
Supplementary material 2 (DOC 10,543 kb)

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Copyright information

© The Botanical Society of Japan and Springer 2011

Authors and Affiliations

  • Haiyong Qu
    • 1
  • Xueting Jiang
    • 1
  • Zebin Shi
    • 2
  • Lianmei Liu
    • 3
  • Shaoling Zhang
    • 1
    Email author
  1. 1.College of HorticultureNanjing Agricultural UniversityNanjingChina
  2. 2.Zhejiang Academy of Agricultural SciencesHangzhouChina
  3. 3.Huaiyin Institute of TechnologyHuaianChina

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